Abstract

Acute megakaryoblastic leukemia (AMKL) is a heterogeneous disease generally associated with poor prognosis. Gene expression profiles indicate the existence of distinct molecular subgroups, and several genetic alterations have been characterized in the past years, including the t(1;22)(p13;q13) and the trisomy 21 associated with GATA1 mutations. However, the majority of patients do not present with known mutations, and the limited access to primary patient leukemic cells impedes the efficient development of novel therapeutic strategies. In this study, using a xenotransplantation approach, we have modeled human pediatric AMKL in immunodeficient mice. Analysis of high-throughput RNA sequencing identified recurrent fusion genes defining new molecular subgroups. One subgroup of patients presented with MLL or NUP98 fusion genes leading to up-regulation of the HOX A cluster genes. A novel CBFA2T3-GLIS2 fusion gene resulting from a cryptic inversion of chromosome 16 was identified in another subgroup of 31% of non-Down syndrome AMKL and strongly associated with a gene expression signature of Hedgehog pathway activation. These molecular data provide useful markers for the diagnosis and follow up of patients. Finally, we show that AMKL xenograft models constitute a relevant in vivo preclinical screening platform to validate the efficacy of novel therapies such as Aurora A kinase inhibitors.

CNS involvement in AMKL. (A) Recipient from AMKL7 cells showing spinal cord–localized tumor indicated by an arrow. (B) FACS analysis of the spinal cord tumor shown in A indicates that it is constituted of megakaryoblastic cells. (C) Histopathological analysis of the spinal cord. Close-up shows a spinal ganglion (inside dashed area) surrounded by leukemic cells. (D) Histopathological analysis of the brain reveals infiltration of the leptomeninges with leukemic cells. Arrow in the main image points to an infiltration of leukemic cells also shown in the top left inset; arrow in the inset points to a cell in mitosis. Bars: (C, left) 5,000 µm; (C [right] and D) 50 µm. (E) MRI image of the AMKL7 patient showing abnormal signals in several spine areas, suggesting leukemia infiltration of the nervous system. Arrow points to an abnormal signal.

The THRAP3-SH3BP2 fusion. (A) Schematic representation of the fusion between THRAP3 (located on chromosome 1) and SH3BP2 (located on chromosome 4). Localization on chromosome and exon–intron gene structure are indicated. Red vertical arrows indicate the targeted introns. Horizontal black arrows indicate localization of the primers used for RT-PCR analysis described in B to detect the fusion transcripts. (B) RT-PCR analysis on a validation cohort of AMKL patients using the primers described in A. Bands appearing in samples 1, 2, and 3 for the THRAP-SH3BP2 fusion were not confirmed by direct sequencing and therefore represent nonspecific amplification. (C) Schematic representation of the THRAP3, SH3BP2, THRAP3-SH3BP2, and SH3BP2-THRAP3 predicted proteins. Red arrows indicate fusion points on each protein.

The CBFA2T3-GLIS2 fusion is recurrent in AMKL. (A) RT-PCR analysis on a validation cohort of AMKL patients using primers located on CBFA2T3 exon 11 and GLIS2 exon 3 (top). Detection of the ARNT transcript was used as an RNA quality control. (B) Schematic representation of the chromosome 16 chromosomal inversion leading to the fusion between CBFA2T3 and GLIS2. Localizations on chromosome and exon–intron gene structures are indicated. Red vertical arrows indicate the introns targeted by the inversion. Horizontal black arrows indicate localization of the primers used for RT-PCR analysis described in C to detect the fusion transcript. (C) RT-PCR analysis of CBFA2T3 and GLIS2 expression in patients with or without the CBFA2T3-GLIS2 fusion. White line indicates that intervening lanes have been spliced out. (D) Western blot analysis of AMKL7 cells using an anti-CBFA2T3 antibody. AMKL7 cell lysates were prepared from fresh cells obtained from tertiary recipients. For controls, 293T cells were transfected with empty or CBFA2T3- or CBFA2T3-GLIS2–encoding vectors. (E) Schematic representation of the CBFA2T3, GLIS2, and CBFA2T3-GLIS2 predicted proteins. Red arrows indicate fusion points on each protein. ZNF, Krüppel-like zinc finger domain.

CBFA2T3-GLIS2 AMKLs exhibit a distinct expression signature. (A) Expression analysis of RNA-seq data showing molecular signature of each sample versus all of the other samples. (B) Genes implicated in the signatures indicated in A were used to perform principal component analysis. (C) Class comparison of AMKL samples presenting the OTT-MAL fusion, CBFA2T3-GLIS2 fusion, acquired trisomy 21, and other alterations. (D) Venn diagram representing common genes between RNA-seq and microarray signatures of AMKL samples presenting CBFA2T3-GLIS2 fusion. (E) Venn diagram representing common genes between RNA-seq and microarray signatures of AMKL samples presenting OTT-MAL fusion. (F) GSEA using a Hedgehog gene list comparing OTT-MAL with CBFA2T3-GLIS2 patients (left) and CBFA2T3-GLIS2 with other non-DS AMKL patients (right). The leading edge genes for each comparison are represented in the bottom panels. FDR, false discovery rate. (G) Selected genes from molecular signatures of the different AMKL subgroups. Asterisk indicates the patient presenting the NUP98-KDM5A fusion. (H) Schematic representation of all fusion genes found in AMKL samples. Hatched squares represent patients with no available material for detection of the novel fusions by bispecific PCR but for which global expression data are available. (I) Flow cytometry analysis of CD56 expression on AMKL7 (CBFA2T3-GLIS2) leukemic blasts (right). Comparison of the CD56 expression between AMKL4 (NUP98-KDM5A) and AMKL7. (J) ChIP analysis using a CBFA2T3-specific antibody or a nonspecific antibody (IgG) on AMKL7 cells or control K562 cells. Quantitative PCR was then performed on immunoprecipitated DNA using primer pairs located in the proximal NCAM1 promoter or in the 3′ UTR. Error bars indicate mean ± SEM.